Taurine supplemented cell culture medium and methods of use

ABSTRACT

The specification describes a composition comprising an improved eukaryotic cell culture medium, which can be used for the production of a protein of interest. Taurine can be added to the serum-free media or chemically-defined media to increase the production of a protein of interest. Methods for recombinantly expressing high levels of protein using the media compositions are included.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/749,037, filed on January 30, 2018, which is a national stageapplication filed under 35 U.S.C. § 371 of International Application No.PCT/US2016/045403, filed Aug. 3, 2016, which claims priority to and thebenefit of U.S. Provisional Patent Application No. 62/200,689, filedAug. 4, 2015, the contents of each of which are herein incorporated byreference in their entireties.

FIELD

The invention relates to medium and methods for the culturing of cellsand for the production of recombinant proteins. The inventionspecifically relates to taurine supplemented medium and methods thereoffor the culturing of recombinant eukaryotic cells for the production ofprotein biotherapeutics.

BACKGROUND

The organic acid taurine, often called a 13-amino acid, is found in highconcentrations in most tissues and is a derivative of the amino acidcysteine (Huxtable, RJ., 1992, Physiol Rev, 72:101-163).

Taurine is present in many tissues of humans and other mammalianspecies, e.g. brain, retina, myocardium, skeletal and smooth muscle,platelets and neutrophils. Taurine is recognized in helpingosmoregulation, membrane stabilization and anti-inflammation, and alsoregulates mitochondrial protein synthesis through enhanced electrontransport chain activity that protects against superoxide generation(Jong et al., 2010, Journal of Biomedical Science 17(Suppl 1):525; Jonget al., 2012, Amino Acids 42:2223- 2232sw). In primary neuronalcultures, taurine has been characterized as a cytoprotectant, due to itssuppression of glutamate-induced toxicity. Various media for embryoculture have been developed containing taurine.

Cell culture techniques comprising amino acid feeds have a long historyof use in the production of recombinant proteins from cultured cells.Amino acids are biosynthetic precursors, energy sources, osmolytes andthe like, and their use in production cultures strongly correlates withcontinuous cell growth and productivity.

However, the physiological events that contribute to productivity andhigh yield protein expression are innumerable, and competing metabolicactivities and transport mechanisms make the design of feedingstrategies a challenge. The type of amino acid supplementation andtiming of addition could also have an impact on the quality of theprotein produced in culture (Altamirano, et al., 2006, Electron. JBiotechnol, 9:61-67). Accumulation of by-products is often problematicin production cell culture, and is considered a consequence ofunbalanced nutrients in cell culture, ultimately inhibiting cell growth(Fan, Y. et al. Biotechnol Bioeng. 2015 Mar; 112(3):521-35). Hypotaurineor an analog or precursor thereof has been suggested in cell culture inorder to achieve the desired results of reduced color intensity of acomposition comprising a recombinantly produced polypeptide(WO2014145098A1, published 13 Sep. 2014) Cell culture medium includingtaurine that promotes the maturation of immature retinal pigmentedepithelium cells into mature retinal pigmented epithelium cells has alsobeen described (WO20131848G9A1. published 12 Dec. 2013). However,optimizing recombinant protein productivity in taurine-supplementedcultures has not been recognized in the art Cell culture processes thatincrease the productivity of the recombinantly expressed proteins, whileminimizing the output of potentially toxic cell metabolism byproducts,such as ammonia, are highly desirable Any consistent gain inproductivity can equate to significantly higher supply at commercialscale of a biotherapeutic product.

Thus there is a need m the art for medium and methods for culturingmammalian cells, wherein the medium allows for healthy and robust cellgrowth and maintenance, and high-titer production of biopharmaceuticaldrug substance.

SUMMARY

The inventors have made the surprising discovery that the inclusion oftaurine in a ceil culture medium increases cellular specificproductivity and allows for lower ammonia byproduct by those cells.Various feeding strategies including taurine permit increased titerprotein production. Furthermore, the addition of taurine has no negativeimpacts on culture performance or resulting antibody quality.

The present invention provides a method for producing therapeuticprotein in high yield comprising culturing a recombinant cell line inmedium containing taurine, wherein the cell line comprises a stablyintegrated nucleic acid encoding the therapeutic protein.

The present invention relates to a cell culture medium, which isserum-free and comprises about 0.1 mM to about 10 mM taurine. Thepresent invention relates to a cell culture medium, which is serum-freeand comprises about 0.1 mM to about 1 mM taurine, about 0.2 to about 1mM taurine, about 0.3 to about 1 mM taurine, about 0.4 to about 1 mMtaurine, or about 0.5 to about 1 mM taurine. The present inventionrelates to a cell culture medium, which is serum-free and comprisesabout 1 mM to about 10 mM taurine. The present invention relates to acell culture medium, which is serum-free and comprises about 1 mM toabout 5 mM taurine, about 1 mM to about 6 mM taurine about 1 mM to about7 mM taurine, about 1 mM to about 8 mM taurine, or about 1 mM to about 9mM taurine

In some embodiments, the medium further comprises additional amino acidsselected from the group consisting of arginine, histidine, lysine,aspartic acid, glutamic acid serine, threonine, asparagine, glutamine,cysteine, glycine, proline, alanine, valine, isoleucine, leucine,methionine, phenylalanine, tyrosine, and tryptophan.

In some embodiments, the medium contains ≤16 9/L hydrolysate. In someembodiments, the medium is free of any hydrolysate.

In one embodiment, the medium contains a base medium that is chemicallydefined, such as a custom formulation or a commercially available basemedium. In one embodiment, the complete medium is chemically defined,free of sera and free of hydrolysate.

In some embodiments, the total process including the base medium andfeeds, contains a total of at least 115 mM of a mixture of ammo adds oramino acid salts. In one embodiment, the mixture of amino adds comprisesamino acids selected from the group consisting of arginine, histidine,lysine, aspartic acid, glutamic acid, serine, threonine, asparagine,glutamine, cysteine, glycine, proline, alanine, valine, isoleucine,leucine, methionine, phenylalanine, tyrosine, and tryptophan, in anamount selected from Table 1.

In some embodiments, the medium contains one or more fatty acids. In oneparticular embodiment, the medium contains a mixture of fatty acids (orfatty acid derivatives) and alpha tocopherol. Fatty acids or fatty acidderivatives are selected from the group consisting of linoleic acid,linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid,arachidic acid, arachidomc acid, lauric acid, behenic acid, decanoicacid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid,and octanoic acid.

In some embodiments, the medium contains a mixture of nucleosides. Inone embodiment, the medium contains adenosine guanosine, cytidine,uridine, thymidine, and hypoxanthine.

In some embodiments, the medium contains a mixture of salts. Saltsinclude divalent cations, such as calcium and magnesium. In oneembodiment, the medium contains calcium chloride and magnesium sulfate.Other salts may include those of phosphate.

In one embodiment, the medium (1) contains 0.1±0.015 mM, 1±0.015 mM,3±0.05 mM. 5±0.10 mM 7±0.15 mM. or 10±0 2 mM taurine. (2) contains≤16g/L of a hydrolysate, (3) is serum-free (4) optionally additionallycontains a mixture of amino acids, (5) contains a mixture of fattyacids. (6) contains a mixture of nucleosides including adenosine,guanosine, cytidine, uridine, thymidine, and hypoxanthine, and (7)contains salts of calcium, magnesium, and phosphate.

The present invention provides a method for producing a protein ofinterest in high yield comprising culturing a recombinant cell line in acell culture medium containing at feast about 0.1 mM to about 10 mMtaurine, wherein the ceil line comprises a stably integrated nucleicacid encoding the protein. In other embodiments, the medium embodies anyof the foregoing aspects of the invention.

In another aspect, the invention provides a method for culturingeukaryotic cells for improved recombinant protein production, comprisingthe steps of; (a) propagating or maintaining cells in a defined cellculture medium during growth phase, (b) supplementing the base cellculture medium with about 0.1 mW to about 10 mM L-taurine and expressinga recombinant protein of interest during production phase, and (c)increasing titer of the protein of interest by the addition of taurine.In some embodiments, the taurine supplement is provided at least onceduring production phase, or twice, three times, four times, or fivetimes during production phase, or on each day for the duration of theproduction phase. In other embodiments, the method further comprisessupplementing the culture medium with about 0.1 mM to about 10 mML-taurine during growth phase. In some embodiments, the method providesimproved production of recombinant protein compared to a eukaryoticcells lacking taurine supplementation, or with less than 0.1 mM taurinesupplementation and under otherwise identical conditions.

In another aspect, the invention provides a method for cultivating cellsin a cell culture medium, such as any embodiment of the medium describedin the foregoing aspect. In one embodiment, the method employs the stepsof propagating or maintaining a cell or cells in a medium that (1)contains taurine at a concentration of at least 0 1 mM±0.015 mM. (2)contains ≤16 g/L hydrolysate, or no hydrolysate (3) is free of sera, (4)and optionally amino acids selected from the group consisting of amixture of ammo acids selected from Table 1.

In one embodiment, the optional mixture of amino acid supplements areselected from the group consisting of the amino acids in Table 1:

TABLE 1 RANGE mM RANGE Amino acid (mmol/L) (g/L) Alanine  0-11.2  0-1Arginine 2.4-11.9  0.5-2.5 Asparagine 1.3-33.3 0.2-5 Aspartic Acid1.5-93.9   0.2-12.5 Cysteine 1.1-19.9  0.2-3 5 Glutamic acid 1.4-47.60.2-7 Glutamine  0-23.9    0-3.5 Glycine  0-16.7   0-1.25 Histidine 1-9.5 0.2-2 Isoleucine 1.5-22.9 0.2-3 Leucine 1.5-38.1 0.2-5 Lysine2.7-24.5  0.5-4.5 Methionine 1.3-13.4 0.2-2 Phenylalanine 1.2-18.2 0.2-3Proline 1.7-26.1 0.2-3 Serine 1.9-57.1 0.2-0 Threonine 1.7-33.6 0.2-4Tryptophan 0.5-14.7 0.1-3 Tyrosine 0.9-22.2 0.2-5 Valine 1.7-34.1 0.2-4

In some embodiments, the ceil or cells are mammalian cells, avian cells,insect cells, yeast cells, or bacteria cells. In one embodiment, thecells are mammalian cells useful in the production of recombinantprotein, such as CHO cells or the derivative CHO-K1. In someembodiments, the cells express a protein of interest such as abiotherapeutic protein. The biotherapeutic protein may be an antigenbinding protein, which may contain an Fc domain. In some embodiments,the protein of interest is an Fc-fusion protein, such as a ScFv moleculeor a trap molecule. Trap molecules include, but are not limited to, theVEGF trap and IL-1 Trap proteins. In some embodiments, the protein ofinterest is an antibody, such as a human monoclonal antibody, humanizedmonoclonal antibody, a bispecific antibody, or an antibody fragment.

Given the positive effects on protein production by including taurine invarious forms of serum-free media, the cells cultured according to thismethod result in an average increase in protein titer. In oneembodiment, when compared to protein titer in a medium that has not beensupplemented with taurine, the cells grown in taurine supplementedculture according to this method produce proteins having a protein titerthat is at least 8% greater than the titer of the comparator controlculture (i.e. culture that has not been supplemented with taurine). Inone embodiment, the cells grown in taurine supplemented culture whencompared to protein titer in media that not been supplemented withtaurine yield a protein titer that is at least 9%, at least 10%, a:least 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, atleast 21%, at least 22%, at least 23%, at least 24%, at least 25%, atleast 26%, at least 27%, at least 28%, or at least 29% greater than thetiter of the comparator control culture.

Likewise, the inclusion of taurine alone in serum-free media allowscultured cells to attain lower ammonia byproduct than without theinclusion of taurine in one serum-free and hydrolysate-free embodimentof the taurine supplemented medium the cell culture is capable ofattaining a reduced ammonia byproduct level (mM NH3) that is at least 4%lower, up to 32% lower than a similar ceil culture in a similar cellculture medium that contains no supplementation (i.e. less than 0.1 mMtaurine or no taurine supplement).

In another embodiment, the method includes the step of adding one ormore point-of-use additions to the cell culture medium. In someembodiments, the point-of-use addition is any one or more of NaHCO₃,glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCl₃, NiSO₄, Na₄EDTA, andNa₃ Citrate. In one embodiment, the method employs the step of addingeach of the following point-of-use chemicals to the cell culture mediumNaHCO₃, glutamine, insulin, glucose, CuSO₄, ZnSO₄, FeCl₃, NiSO₄,Na₄EDTA, and Na₃ Citrate. In some embodiments, the point-of-useadditions can be included in the medium at the outset.

In a specific embodiment, the aspect provides a method for cultivatingcells in a serum-free medium consisting essentially of (1) taurine at aconcentration of at least 0.1 mM; (2) contains ≤16 g/L of a hydrolysate,(3) is serum-free and (4) optionally additionally contains at leastabout 20 mM, or at least about 25 mM, or at least about 30 mM, or atleast about 40 mM, or at least about 50 mM, or at least about 60 mM, orat least about 70 mM total of a mixture of ammo acids selected from thegroup consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In another aspect, the invention provides a method for producing aprotein of interest by employing the steps of (1) introducing into acell a nucleic acid sequence that encodes a protein of interest; (2)selecting a cell or cells expressing the protein of interest. (3)culturing the selected cell in an embodiment of the serum-free cellculture medium described in any preceding aspect or according to anyembodiment of the method described herein; and (4) expressing theprotein of interest in the cell, wherein the protein of interest issecreted into the medium in some embodiments, the cell used in theproduction of the protein is a mammalian cell capable of producing abiotherapeutic, such as CHO, 293, and BHK cell, or any derivatives ofthem In one embodiment, the cell is a CHO cell, such as a CHO-K1 cell.

In some embodiments the protein of interest is an antigen bindingprotein In some embodiments, the protein of interest is a protein thathas an Fc domain. In some cases, those two proteins of interest may overap. such as in the case of a receptor-Fc-fusion protein, an antibody,and a ScFv protein for example. Thus, in some embodiments, the proteinof interest is an antibody such as a human antibody or a humanizedantibody, an antibody fragment, such as an Fab or F(ab′)₂, a bispecificantibody, a trap molecule, such as a VEGF-Trap or an IL-1-Trap. an ScFvmolecule a soluble TCR-Fc fusion protein, or the like.

In one embodiment, the protein of interest is capable of being producedat an average 14, 15, 16, or 17 day titer that is at least 8% greaterthan the average 14, 15, 16 or 17 day titer produced by a similar cellin a serum-free cell culture medium that contains less than 0.1 mM or notaurine supplementation, in one embodiment, the protein of interest iscapable of being produced at an average 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 or 17 day titer that is at least 9%, at least 10%, at least 11%,at feast 12%, at least 13%, at least 14%, at least 15%, at least 16%, atleast 17%, at least 18%, at least 19%, at least 20%, at least 21%, atleast 22%, at least 23%, at least 24%, at least 25%, at least 26%, atleast 27%, at least 28%, or at least 29% greater than the average 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 day titer produced by a similarcell in a serum free cell culture medium that contains less than 0.1 mMor no taurine supplementation.

In another embodiment, the protein of interest is produced by (1)introducing into a CHO cell a nucleic acid sequence that encodes aprotein of interest, such as an antibody or other antigen-bindingprotein; (2) selecting a cell stably expressing the protein of interest;(3) culturing the selected cell in a serum-free cell culture mediumcomprising about 0.1 mM to about 10 mM taurine.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figure shows the protein titer (yield) from samples retrieved ateach day of production culture in an Ab3-producing cell culture wheretaurine-supplementation is provided (solid squares connected by solidlines) compared to no taurine supplementation (x connected by dottedlines). The benefits of taurine-supplemented culture to protein yieldcan be seen as early as day 6 of the production culture.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to particularmethods and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention is defined by the claims.

As used in this specification and the appended claims the singular forms“a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus tor example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the aftupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of the present invention, particular methods and materialsare now described. All publications mentioned herein are incorporatedherein by reference in their entirety.

The applicants have made the surprising discovery that the addition oftaurine to a cell culture medium improves protein production by arecombinant cell in a cell culture relative to a cell culture mediumthat contains very little or no taurine.

Before the present cell cultures and methods are described, it is to beunderstood that this invention is not limited to particular methods andexperimental conditions described, as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.The methods and techniques described herein are generally performedaccording to conventional methods known in the art and as described invarious general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual. 3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001) and Ausubel et al., Current Protocols in MolecularBiology. Greene Publishing Associates (1992), Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), and Julio E Celis, Ceil Biology: ALaboratory Handbook, 2^(nd) ed., Academic Press New York. N.Y. (1998),and Dieffenbach and Dvekster, PCR Primer. A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1995). Allpublications mentioned throughout this disclosure are incorporatedherein by reference in their entirety.

Definitions

Taurine is also known as 2-aminoethanesulfonic acid (IUPAC nomenclature;CAS Registry No. 107-35-7). “Taurine” and “L-taurine” are usedinterchangeably to refer to the same organic compound. Taurine is anorganic acid containing an amino group, however is not considered an“amino acid” as traditionally known to those in the art, whereas aminoacids contain both an amino group and a carboxyl group. Biosynthesis oftaurine occurs when hypotaurine, which is a derivative of cysteine, isconverted to taurine by oxidation.

The terms “supplementation”, “supplementing”, “supplemented with”, andthe like, refer to adding an ingredient, a component, a molecule, etc.which may be used in a medium for cell culture to maintain and/orpromote the growth and/or differentiation of cells, to extend orstrengthen an attribute of the culture or cells as a whole, or to makeup for a deficiency. To this end, taurine-supplementation includes theaddition of taurine at a particular concentration in a solution to theculture medium.

The terms “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by a peptide bond.Peptides, polypeptides and proteins may also include modifications suchas glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, alkylation, hydroxylation and ADP-ribosylation.Peptides, polypeptides, and proteins can be of scientific or commercialinterest, including protein-based drugs. Peptides, polypeptides, andproteins include, among other things, antibodies and chimeric or fusionproteins. Peptides, polypeptides, and proteins are produced byrecombinant animal cell lines using cell culture methods.

The term “heterologous polynucleotide sequence”, as used herein refersto nucleic acid polymers encoding proteins of interest, such as chimericproteins (like trap molecules), antibodies or antibody portions (e.g.,VH, VL, CDR3) that are produced as a biopharmaceutical drug substance.The heterologous polynucleotide sequence may be manufactured by geneticengineering techniques (e.g., such as a sequence encoding a chimericprotein, or a codon-optimized sequence, an intronless sequence etcetera) and introduced into the cell, where it may reside as an episomeor be intergrated into the genome of the cell. The heterologouspolynucleotide sequence may be a naturally occurring sequence that isintroduced into an ectopic site within the production cell genome. Theheterologous polypeptide sequence may be a naturally occurring sequencefrom another organism, such as a sequence encoding a human ortholog.

“Antibody” refers to an immunoglobulin molecule consisting of fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain has a heavy chainvariable region (HCVR or VH) and a heavy chain constant region. Theheavy chain constant region contains three domains, CH1, CH2 and CH3.Each light chain has a light chain variable region and a light chainconstant region. The light chain constant region consists of one domain(CL). The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody” includesreference to both glycosylated and non-glycosylated immunoglobulins ofany isotype or subclass. The term “antibody” includes antibody moleculesprepared, expressed, created or isolated by recombinant means, such asantibodies isolated from a host cell transfected to express theantibody. The term antibody also includes bispecific antibody, whichincludes a heterotetrameric immunoglobulin that can bind to more thanone different epitope. Bispecific antibodies are generally described inUS Patent Application Publication No 2010/0331527, which is incorporatedby reference into this application.

The term “antigen-binding portion” of an antibody (or “antibodyfragment”), refers to one or more fragments of an antibody that retainthe ability to specifically bind to an antigen. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward el al. (1989) Nature241:544-546), which consists of a VH domain, (vi) an isolated CDR, and(vii) an scFv, which consists of the two domains of the Fv fragment, VLand VH, joined by a synthetic linker to form a single protein chain inwhich the VL and VH regions pair to form monovalent molecules. Otherforms of single chain antibodies, such as diabodies are also encompassedunder the term “antibody” (see e g Holliger et at. (1993) PNAS USA90:6444-6448. Poljak et at. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies andHybridomas 6:93-101) and use of a cysteine residue, a marker peptide anda C-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipnyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibodyportions, such as Fab and F(ab′)2 fragments, can be prepared from wholeantibodies using conventional techniques, such as via papain or pepsindigestion of whole antibodies. Moreover, antibodies, antibody portionsand immunoadhesion molecules can be obtained using standard recombinantDNA techniques commonly known in the art (see Sambrook et al., 1989).

The term “human antibody” is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs and in particularCDR3. However, the term “human antibody”, as used herein, is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepares, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library,antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.“Fc fusion proteins” comprise part or all of two or more proteins, oneof which is an Fc portion of an immunoglobulin molecule, which are nototherwise found together in nature. Preparation of fusion proteinscomprising certain heterologous polypeptides fused to various portionsof antibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al., Proc. Natl. Acad ScL USA 88:10535. 1991; Byrn et al., Nature 344:677, 1990. and Hollenbaugh et al.,“Construction of Immunoglobulin Fusion Proteins”, in Current Protocolsin Immunology, Suppl 4. pages 10.19.1-10.19.11, 1992. “Receptor Fcfusion proteins” compose one or more extracellular domain(s) of areceptor coupled to an Fc moiety, which in some embodiments comprises ahinge region followed by a CH2 and CH3 domain of an immunoglobulin. Insome embodiments, the Fc-fusion protein contains two or more distinctreceptor chains that bind to a one or more ligand(s). For example, anFc-fusion protein is a trap, such as for example an IL-1 trap (e.g.rilonacept, which contains the IL-1RAcP ligand binding region fused tothe IL-1R1 extracellular region fused to Fc of higG1: see U.S. Pat. No.6.927,004). or a VEGF trap (e.g. aflibercept which contains the lgdomain 2 of the VEGF receptor Flt1 fused to the lg domain 3 of the VEGFreceptor Flk1 fused to Fc of hlgG1; see U.S. Pat. Nos. 7,087,411 and7,279,159).

Cell Culture

The terms “cell culture medium” and “culture medium” refer to a nutrientsolution used for growing mammalian cells that typically provides thenecessary nutrients to enhance growth of the cells, such as acarbohydrate energy source, essential (e.g. phenylalanine, valine,threonine, tryptophan methionine, leucine, isoleucine, lysine, andhistidine) and nonessential (e.g. alanine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, proline, serine, andtyrosine) amino acids, trace elements, energy sources, lipids, vitamins,etc. Cell culture medium may contain extracts, e.g. serum or peptones(hydrolysates), which supply raw materials that support cell growth.Media may contain yeast-derived or soy extracts instead ofanimal-derived extracts. Chemically defined medium refers to a cellculture medium in which all of the chemical components are known (i.e.have a known chemical structure). Chemically defined medium is entirelyfree of animal-derived components, such as serum- or animal-derivedpeptones. In one embodiment, the medium is a chemically defined medium.

The solution may also contain components that enhance growth and/orsurvival above the minimal rate, including hormones and growth factors.The solution is preferably formulated to a pH and salt concentrationoptimal for cell survival and proliferation.

A “cell line” refers to a cell or cells that are derived from aparticular lineage through serial passaging or subculturing of cells.The term “cells” is used interchangeably with “cell population”.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of eukaryotes,such as non-human animal cells, mammalian cells, human cells, aviancells, insect cells, yeast cells, or cell fusions such as, for example,hybridomas or quadromas. In certain embodiments, the cell is a human,monkey, ape, hamster, rat or mouse cell. In other embodiments, the cellis selected from the following cells. CHO (e.g. CHC K1, DXB-11 CHO,Veggie-CHO), COS (e.g. COS-7), retinal cell. Vero, CV1, kidney (e.g.HEK293,293 EBNA, MSR 293, MDCK, HaK, BHK21), Hela, HepG2, WI38, MRC 5,Colo25, HB 8065, HL-60, lymphocyte, e.g. Jurkat (T lymphocyte) or Daudi(B lymphocyte). A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT cell, stem cell, tumor cell, and a cell line derivedfrom an aforementioned cell. In some embodiments, the cell comprises oneor more viral genes, e.g. a retinal cell that expresses a viral gene(e.g. a PER.C6® cell). In some embodiments, the cell is a CHO cell. Inother embodiments, the cell is a CHO K1 cell.

One aspect of the invention relates to a seed culture in which a cellpopulation is expanded prior to protein production and harvest in theproduction culture. Taurine may be added to the base medium in a seedculture formulation, according with the invention as described herein.

Another aspect of the invention relates to a production culture in whichprotein is produced and harvested. Prior to production phase, there istypically a growth phase (also known as a seed train or seed culture)wherein all components for cell culturing are supplied to the culturingvessel at the start of the culturing process then cell population isexpanded until ready for production scale. As such, the culturing vesselis inoculated with cells at a suitable seeding density for the initialcell growth phase depending on the starting cell line. In some aspects,taurine may be added to the basal culture medium in a seed cultureformulation, according with the invention as described herein in orderto further improve or enhance the productivity of the cells in thesubsequent production phase.

One aspect of the invention relates to a production culture wherein cellculture conditions are modified to enhance the growth of recombinanteukaryotic cells while improving the production of one or morerecombinant proteins of interest by such cells and maintaining cellviability, in particular by adding taurine to the production culturemedium and/or the seed tram culture. In the production culturing vesselor bioreactor, a basal culture medium and cells are supplied to aculturing vessel following a seed culture or growth phase. In certainembodiments the cell supernatant or cell lysate is harvested followingthe production culture. In other embodiments, the polypeptide or proteinof interest is recovered from the culture medium or cell lysate, orwhatever the case may be depending on the location of the protein ofinterest, using techniques well known in the art.

Culturing vessels include, but are not limited to well plates, T-flasks,shake flasks, stirred vessels, spinner flasks, hollow fiber, air liftbioreactors, and the like. A suitable cell culturing vessel is abioreactor. A bioreactor refers to any culturing vessel that ismanufactured or engineered to manipulate or control environmentalconditions. Such culturing vessels are well known in the art.

Bioreactor processes and systems have been developed to optimize gasexchange, to supply sufficient oxygen to sustain cell growth andproductivity and to remove CO₂. Maintaining the efficiency of gasexchange is an important criterion for ensuring successful scale up ofcell culture and protein production. Such systems are well-known to theperson having skill in the art.

In the polypeptide production phase, a “fed-batch cell culture” or“fed-batch culture” refers to a batch culture wherein the animal cellsand culture medium are supplied to the culturing vessel initially andadditional culture nutrients are slowly fed, continuously or in discreteincrements, to the culture during culturing, with or without periodiccell and/or product harvest before termination of culture. Fed-batchculture includes “semi-continuous fed-batch culture” whereinperiodically whole culture (which may include cells and medium) isremoved and replaced by fresh medium. Fed-batch culture is distinguishedfrom simple “batch culture” whereas all components for cell culturing(including the animal cells and all culture nutrients) are supplied tothe culturing vessel at the start of the culturing process in batchculture. Fed-batch culture can be further distinguished from perfusionculturing insofar as the supernatant is not removed from the culturingvessel during the process, whereas in perfusion culturing the cells arerestrained in the culture by. e.g., filtration, and the culture mediumis continuously or intermittently introduced and removed from theculturing vessel. However, removal of samples for testing purposesduring fed-batch cell culture is contemplated. The fed-batch processcontinues until it is determined that maximum working volume and/orprotein production is reached.

The phrase “continuous cell culture” when used herein relates to atechnique used to grow cells continually, usually in a particular growthphase. For example, if a constant supply of cells is required, or theproduction of a particular polypeptide or protein of interest isrequired, the cell culture may require maintenance in a particular phaseof growth. Thus, the conditions must be continually monitored andadjusted accordingly in order to maintain the cells in that particularphase.

Media

The present invention provides a cell culture medium, which isserum-free, comprising about 0.1 mM to 10 mM taurine. “Serum-free”applies to a cell culture medium that does not contain animal sera, suchas fetal bovine serum. The serum-free media may contain≤16 g/L ofhydrolysates, such as soy hydrolysate. The present invention alsoprovides chemically defined media, which is not only serum-free, butalso hydrolysate-free. “Hydrolysate-free” applies to cell culture mediathat contains no exogenous protein hydrolysates such as animal or plantprotein hydrolysates such, for example peptones, tryptones and the tike.“Base medium” is the initial medium (present in the seed train and/or atday 0 of the cell culture production) in which the cells are propagatedand contains all the necessary nutrients, which includes a base mixtureof amino acids. Various recipes (i.e. formulations) for base media maybe manufactured or purchased in commercially available lots Likewise“base feed medium” contains mixtures of supplemental nutrients that arecommonly consumed during a production culture and are utilized in afeeding strategy (for a so-called “fed-batch” culture). Varieties ofbase feed media are commercially available. A “feed” includes scheduledadditions or additions to media at regular intervals such as accordingto a protocol, including a continuous feed culture system, as in achemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec;17(6): 1032-41). or according to a fed-batch process (Y. M. Huang etal., Biotechnol Prog. 2010 Sep-Oct:26(5): 1400-10). For example, aculture may be fed once per day, every other day, every three days, ormay be fed when the concentration of a specific medium component, whichis being monitored, falls outside a desired range.

The elimination of serum and reducing or eliminating hydrolysales fromcell culture media, while reducing lot-to-lot variability and enhancingdownstream processing steps, unfortunately diminishes cell growth,viability and protein expression. Thus, chemically defined serum-freeand low to no hydrolysate media requires additional ingredients toimprove cell growth and protein production.

Thus, the cell culture medium of the invention composes a base mediumcontaining all necessary nutrients for a viable cell culture. Taurinemay be added to the base medium in a seed culture formulation, accordingwith the invention as described herein. Furthermore, taurine may beadded to the base medium in a production culture formulation, which maythen be fed periodically (as in so-called “fed-batch” cultures) with orwithout additional ingredients such as polyamines or increasedconcentrations of components like amino acids, salts, sugars, vitamins,hormones, growth factors, buffers, antibiotics, lipids, trace elementsand the like depending on the requirements of the cells to be culturedor the desired cell culture parameters.

The invention provides that the taurine-supplemented cell culture mediummay be depleted of amino acids over the course of the protein productionculture, where no additional amino acid supplementation is provided, orthe taurine-supplemented cell culture medium may be “non-depleted”,where amino acid supplementation is provided for the depleted aminoacids (as described below). The inventors have observed that culturessupplemented during production phase with taurine improve recombinantprotein production under various culture conditions as described in theforegoing.

The invention provides taurine-supplemented medium which containstaurine at a concentration (expressed in millimoles per liter) of atleast about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 mM.

In one embodiment, the medium additionally contains 100 μM±15 μMornithine, or 300 μM±45 μM ornithine, or 600 μM±90 μM ornithine, or even900 μM±135 μM ornithine in another embodiment, the medium contains atleast about 5 mg/L±1 mg/L ornithine·HCl, or at least about 10 mg/L±12mg/L ornithine·HCl, 15 mg/L±2.25 mg/L ornithine·HCl, or at least about50 mg/L±7.5 mg/L ornithine·HCl, or at least about 100 mg/L±15 mg/Lornithine·HCl, or at least about 150 mg/L±22.5 mg/L ornithine•HCl.

Putrescine may optionally be added to the supplemented media. Putrescinehas been included, at very low concentrations, as a component in somecell culture media formulations; see for example WO 2005/028626. whichdescribes 0.02-0.08 mg/L putrescine. U.S. Pat. No. 5.426,699 (0.08mg/L): U.S. Pat. No. R,E30,985 (0.16 mg/L): U.S. Pat. No. 5,811,299(0.27 mg/L). U.S. Pat. No. 5,122,469 (0.5635 mg/L); U.S. Pat. No.5,063,157 (1 mg/L); WO 2008/154014 (˜100 μM−˜1000 μM); US Pat. App No.2007/0212770 (0.5-30 mg/L polyamine; 2 mg/L putrescine: 2 mg/Lputrescine+2 mg/L ornithine; 2 mg/L putrescine+10 mg/L ornithine).

In some embodiments, the media is further supplemented with acombination of ornithine and putrescine. wherein the putrescine can beat a concentration of at least about 150 to 720 μM. In some embodiments,the media is further supplemented with putrescine at a concentration ofabout 170 to 230 μM. In one embodiment, the medium contains 200 μM±30 μMputrescine in addition to ≥90 μM±15 μM ornithine. In one embodiment, themedium contains≤30 mg/L±4.5 mg/L putrescine·2HCl in addition to ≤15mg/L±2 25 mg/L ornithine. In another embodiment, the medium contains ≥30mg/L±4.5 mg/L putrescine·2HCl in addition to ≥15 mg/L±2.25 mg/Lornithine·HCl. (See International Publication No. WO2014/144198A1,published on Sep. 18, 2014, which is herein incorporated by reference inits entirety.

In still other embodiments, ornithine is present in the medium at aconcentration ranging from 0.09±0.014 mM to 0.9±0.14 mM, such as0.09±0.014 mM, 0.3±10.05 mM. 0.6±0.09 mM. or 0.9±0.14 mM ornithine. Insome embodiments, the medium also contains at least 0.20±0.03 mMputrescine. In some embodiments, the additional putrescine is at aconcentration ranging from 0.20±0.03 mM to 0.714±0.11 mM, such as0.20±0.03 mM. 0.35±0.06. or 0.714±0.11 mM putrescine.

Various other supplements may be added to the culture medium, and arewithin the skill of the person in the art to determine additionallyappropriate conditions. In some embodiments, the medium is supplementedwith a mixture of amino acids selected from the group consisting ofaspartic acid, cysteine, glutamic acid, glycine, lysine, phenylalanine,proline, serine, threonine, valine, arginine, histidine, asparagine,glutamine, alanine isoleucine, leucine, methionine, tyroshe, andtryptophan, in order to be non-depleted or as supplemental nutrients areneeded.

In one embodiment, the media s further supplemented with about 170 μM to175 μM nucleosides. In one embodiment, the media contains purinederivatives in a cumulative concentration of at least 40 μM, at least 45μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, atleast 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90μM, at least 95 μM, at least 100 μM, or at least 105 μM. In oneembodiment, the media contains about 100 μM to 110 μM of purinederivatives. Purine derivatives include hypoxanthine and the nucleosidesadenosine and guanosine. In one embodiment, the media containspyrimidine derivatives in a cumulative concentration of at least 30 μM,at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least55 μM, at least 60 μM, or at least 66 μM. In one embodiment, the mediacontains about 65 μM to 75 μM of pyrimidine derivatives. Pyrimidinederivatives include the nucleosides thymidine, uridine, and cytidine. Inone particular embodiment, the media contains adenosine, guanosine,cytidine, uridine, thymidine and hypoxanthine.

In addition to the inclusion of any of the above additives, in oneembodiment, the media is further supplemented with micromolar amounts offatty acids (or fatty acid derivatives) and tocopherol. In oneembodiment, the fatty acids include any one or more of linoleic acid,linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid,arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoicacid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid,and octanoic acid. In one embodiment, the media contains tocopherol,linoleic acid, and thioctic acid.

In one embodiment, the media also may be further supplemented with amixture of vitamins, which includes other nutrients and essentialnutrients, at a cumulative concentration of at least about 700 μM or atleast about 2 mM. In one embodiment, the mixture of vitamins containsone or more of D-biotin, choline chloride, folic acid, myo-inositol,niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin,thiamine HCl, vitamin B12, and the like. In one embodiment, the mixtureof vitamins includes all of D-biotin, choline chloride, folic acid,myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa),riboflavin, thiamine HCl. and vitamin B12.

Various embodiments of the media of the invention include any of thecombinations of the above described embodiments, including chemicallydefined, hydrolysate-free serum-free media comprising taurine in theindicated amounts, plus inter alia (a) amino acids; (b) optionallynucleosides; (c) salts of divalent cations; (d) fatty acids andtocopherol; and (e) vitamins. In some embodiments, all small amounts ofhydrolysates may be added to the taurine-supplemented media.

The applicants envision that in the practice of this invention any oneor more of a variety of base media or combinations thereof, to which thetaurine may be used Base media are generally known m the art and includeinter aha Eagle's MEME (minimal essential media) (Eagle, Science. 1955,112(3168):501-504), Ham's F12 (Ham, Proc Nat'l. Acad Sci. USA, 1965.53:288-293), F-12 K medium, Duibecco's medium, Dulbecco's Modified EagleMedium (Proc. Natl. Acad. Sci. USA., 1952 August: 38(8): 747-752),DMEM/Ham's F12 1:1, Trowell's T8, A2 media Holmes and Wolf, Biophys.Biochem. Cytol., 1961, 10:389-401), Waymouth media (Davidson andWaymouth, Biochem. J., 1945, 39(2):188-199), Williams E media (William'set al., Exp. Cell Res., 1971, 69:105 et seq.), RPMI 1640 (Moore et al.,J. Amer. Med Assoc., 1967, 199:519-524) MCDB 104/110 media (Bettger etal., Proc Nat'l. Acad. Sci. USA, 1981. 78(9):5588-5592). Ventrex HL-1media, albumin-giobulin media (Orr et al., Appl. Microbiol., 1973,25(1):49-54). RPMI-1640 Medium. RPMI-1641 Medium, Iscove's ModifiedDulbecco's Medium, McCoy's 5 A Medium. Leibovitz's L-15 Medium, andserum-free media such as EX-CELL™ 300 Series (JRH Biosciences, Lenexa,Kansas), protamine-zinc-insulin media (Weiss et al., 1974, U.S. Pat. No.4,072,565), biotin-folate media (Cartaya, 1978, US Re30.985).Transferrin-fatty acid media (Baker, 1982. U.S. Pat. No. 4,560,655),transferrin-EGF media (Hasegawa, 1982. U.S. Pat. No. 4,615,977.Chessebeuf. 1984. U.S. Pat. No. 4,786,599). and other media permutations(see Inlow, U.S. Pat. No. 3,048.728; Drapeau. U.S. Pat. No. 7,294,484;Mather. U.S. Pat. No. 5,122,469; Fuaikawa. U.S. Pat. No. 5,976,833.Chen. U.S. Pat. No. 6,180.401; Chen. U.S. Pat. No. 5.856,179.Etcheverry, U.S. Pat. No. 5,705,364; Etcheverry, U.S. Pat. No.7,666,416; Ryll, U.S. Pat. No. 6,528,286; Singh, U.S. Pat. No.6,924,124; Luan, U.S. Pat. No. 7,429,491. and the like).

In a particular embodiment, the media is chemically defined and containsin addition to the taurine, amino acid mixtures as defined herein, CaCl₂2H₂ O; HEPES buffer, KCl; MgSO₄; NaCl; Na₂HPO₄ or other phosphate salts;pyruvate; D-biotin; choline chloride; folic acid; myo-inositol;niacinamide; pyridoxine HCl; D-pantothenic acid: riboflavin; thiamineHCl; vitamin B12; p-aminobenzoic acid, ethanofamine HCl; poloxamer 188;DL-a-tocopherol phosphate; linoleic acid: Na₂SeO₃; thioctic acid; andglucose; and optionally adenosine; guanosine; cytidine; uridine;thymidine, and hypoxanthine 2Na.

In one embodiment, the starting osmolarity of the media of the inventionis 200-500, 250-400, 275-350, or about 300 mOsm. During growth of thecells in the media of the invention, and in particular following anyfeedings according to a fed batch protocol, the osmolarity of theculture may increase up to about 350, 400, 450, 500 or up to about 550mOsm.

In some embodiments wherein the osmolarity of the defined medium is lessthan about 300, the osmolarity is brought to about 300 with the additionof one or more salts in excess of the amount specified. In oneembodiment, osmolarityis increased to a desired level by adding one ormore of an osmolyte selected from sodium chloride, potassium chloride, amagnesium salt, a calcium salt, an amino acid salt, a salt of a fattyacid, sodium bicarbonate, sodium carbonate, potassium carbonate, achelator that is a salt, a sugar (e.g., galactose, glucose, sucrose,fructose, fucose, etc.), and a combination thereof. In one embodiment,the osmolyte is added over and above its concentration in a componentalready present in the defined medium (e.g., a sugar is added over andabove the concentration specified for a sugar component).

Each and every embodiment of the media described above, as well as anyother serum-free media containing at least about 0.1 mM taurine isreferred to as taurine supplemented media. Conversely, media containingno taurine, or media containing less than 0.1 mM taurine are hereinafterreferred to as non-taurine supplemented media or no taurinesupplementation.

Fed-Batch Culture

Feeding strategies for cell culture aim to ensure the optimal growth andpropagation of cells outside of a multicellular organism or tissue.Suitable culture conditions for mammalian cells are known in the art.See e.g. Animal cell culture; A Practical Approach, D. Rickwood. ed.,Oxford University Press. New York (1992). Mammalian cells may becultured in suspension or while attached to a solid substrate. Fluidizedbed bioreactors, hollow fiber bioreactors, rotter bottles, shake flasks,or stirred tank bioreactors, with or without microcarriers, and operatedin a batch, fed batch, continuous, semi-continuous, or perfusion modeare available for mammalian cell culture. Cell culture media orconcentrated feed media may be added to the culture continuously or atintervals during the culture. For example, a culture may be fed once perday, every other day, every three days, or may be fed when theconcentration of a specific medium component, which is being monitored,falls outside a desired range.

In addition to the inclusion of taurine, in one embodiment media may befurther supplemented with amino acids in a cumulative (total)concentration of at least 20 mM. In one embodiment, the concentration ofinitial amino acids included in the starting cell culture medium is notincluded in such cumulative (total) concentration of supplemented aminoacids. In one embodiment of the cell culture medium, or in the method toculture cells or the method to produce a protein of interest, the mediamay be supplemented in an amount greater than about 20 mM, greater thanabout 25 mM, greater than about 30 mM, greater than about 40 mM greaterthan about 50 mM, or greater than about 60 mM, greater than about 70 mM,greater than about 100 mM, greater than about 200 mM, greater than about300 mM, greater than about 400 mM, or greater than about 500 mM See alsoTable 1 herein. In one embodiment the amount of amino acids added to themedia is about 30 mM±10 mM or more.

Supplemental feeding regimens may be optimized by those skilled in theart to support cell growth, minimize cell stress or to provide a“non-depleted medium” during production phase.

“Non-depleted medium” includes cell culture medium that has beendetermined to have the nutrients, in particular, the amino acidsnecessary for production of a recombinant protein of interest. Aminoacid feeds typically supplement the amino acids needed as buildingblocks for producing a recombinant protein in a cell culture. However,some amino acids may be depleted faster than others depending on therequirements of that particular protein produced by the cells inculture. In an non-depleted medium, the feeding regime has beendetermined such that necessary amino acids are replenished as they areconsumed. Thus, depletion and subsequently optimal consumption rates(pg/cell day) may be determined by the following steps culturingeukaryotic cell(s) expressing the protein of interest in a cell culturemedium; measuring each amino acid concentration in the culture medium attime points to establish a depletion level; identifying the depletiontime point at which the amino acid concentration falls below thedepletion level, calculating consumption rates for each amino acid anddetermining the optimal consumption rate as the consumption rate at thetime point immediately prior to the depletion time point. The cellculture is then supplemented with an appropriate concentration of aparticular amino acid to maintain such optimal consumption rates asdetermined. In order for the culture medium to be non-depleted.

It is understood that the present invention provides ataurine-supplemented cell culture medium that improves protein titer indepleted, as well as non-depleted, cultures.

The present invention provides a cell culture comprising a cell lineexpressing a protein of interest in a taurine-supplemented medium asdescribed above. Examples of cell lines that are routinely used toproduce protein biotherapeutics include infer alia primary cells. BSCceils, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells,VERO cells, MDBK cells, MDCK cells, CRFK cells. RAF cells, RK cells,TCMK-1 cells, LLCPK cells. PK15 ceils LLC-RK cells, MDOK cells, BHKcells. BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRG-5 cells,WI-38 cells, 3T3 cells, 293 cells, Per C6 cells and chicken embryocells. In one embodiment, the cell line is a CHO cell line or one ormore of several specific CHO cell variants optimized for large-scaleprotein production, e.g., CHO-K1.

In one embodiment, the taurine-supplemented cell culture containsinsulin, which can be added as a point-of-use ingredient to the media orcan be included in the media formulation. In one embodiment, the cellline comprises cells capable of producing a biotherapeutic protein.

In one embodiment, the media is supplemented at intervals during cellculture according to a fed-batch process. Fed-batch culturing isgenerally known in the art and employed to optimize protein production(see Y. M. Huang et al., Biotechnol Prog 2010 Sep-Oct:26(5): 1400-10)

The cell growth phase or seed culture (i.e. a first cell culture) whereno exchange of medium is provided, is typically followed by a distinctsecond culture, known as the polypeptide production phase. Fed-batchprocesses are typically used during the production phase.

The invention provides a cell culture medium composing about 0.1 mM toabout 10 mM taurine at the start of production cell culture (day 0).Alternatively, cell culture medium comprising about 0.1 mM to about 10mM taurine may be supplemented on day 1, day 2, day 3, day 4, day 5, day6, day 7 day 8, day 9, and/or day 10 of the production cell culture. Thecell culture medium added to the production culture on multiple dayscomprises a total amount of taurine between about 0.1 mM to about 10 mM.The cell culture medium composing total taurine between about 0.1 mM toabout 10 mM may be added in any sequential manner.

Taurine may also be supplemented in the basal medium in the seed trainexpansion phase.

Supplemental feed may be performed to include additional nutrients, suchas vitamins, amino acids and other nutrients as described hereinabove,at intervals at a frequency of every day, or every 2-3 days, for theduration of the production culture. Supplemented feed may be performed(supplemented media containing nutrients are added) at least 2 times, orat least 8 times, throughout the duration of the production culture fora 2 week or more culture. In another embodiment, the supplemental feedcould be performed on each day for the duration of the culture.Alternative culture feeding schedules are also envisioned.

Additional amino acid supplementation may also be performed to provide anon-depleted medium, wherein depleted amino acids are determinedaccording to methods known in the art and described herein. When thisregime is employed, additional amino acids are supplemented or added atintervals, preferably at a frequency of every day, or every 2-3 days,for the duration of the production culture depending on thedetermination of amino acid depletion In one embodiment, the mixture ofadditional amino acids to maintain a non-depleted ceil culture medium isadded to the culture on or about day 1, on or about day 2, on or aboutday 3, on or about day 4, on or about day 5, on or about day 6, on orabout day 7, on or about day 8, on or about day 9, on or about day 10,on or about day 11, on or about day 12, on or about day 13, and on orabout day 14, for a 2 week or more culture. Alternative culture feedingschedules are also envisioned.

Animal cells, such as CHO cells, may be cultured in small scalecultures, such as in 125 ml containers having about 25 mL of media, 250ml containers having about 50 to 100 mL of media, 500 mL containershaving about 100 to 200 mL of media. Alternatively, the cultures can belarge scale such as for example 1000 mL containers having about 300 to1000 mL of media, 3000 mL containers having about 500 mL to 3000 mL ofmedia. 8000 mL containers having about 2000 mL to 8000 mL of media, and15000 mL containers having about 4000 mL to 15000 mL of media. Culturesfor manufacturing can contain 10,000 L of media or more. Large scalecell cultures, such as for clinical manufacturing of proteintherapeutics, are typically maintained for days, or even weeks, whilethe cells produce the desired protein(s). During this time the culturecan be supplemented with a concentrated feed medium containingcomponents, such as nutrients and amino acids, which are consumed duringthe course of the culture. Concentrated feed medium may be based on anycell culture media formulation. Such a concentrated feed medium cancontain most of the components of the cell culture medium at, forexample, about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X,100X, 200X, 400X, 600X, 800X, or even about 1000X of their normal usefulamount. Concentrated feed media are often used in fed batch cultureprocesses.

In some embodiments, the cell culture containing taurine is furthersupplemented with “point-of-use additions”, also known as additions,point-of-use ingredients, or point-of-use chemicals, during the courseof cell growth or protein production Point-of-use additions include anyone or more of a growth factor or other proteins, a buffer, an energysource, a salt, an amino acid, a metal, and a chelator. Other proteinsinclude transferrin and albumin. Growth factors, which include cytokinesand chemokines, are generally known in the art and are known tostimulate cell growth, or in some cases, cellular differentiation. Agrowth factor is usually a protein (e.g., insulin), a small peptide, ora steroid hormone, such as estrogen DHEA, testosterone, and the like. Insome cases, a growth factor may be a non-natural chemical that promotescell proliferation or protein production, such as e.g., tetrahydrofolate(THF), methotrexate, and the like. Non-limiting examples of protein andpeptide growth factors include angiopoietins, bone morphogeneticproteins (BMPs), brain-derived neurotrophic factor (BDNF), epidermalgrowth factor (EGF), erythropoietin (EPO), fibroblast growth factor(FGF), glial cell line-derived neurotrophic factor (GDNF), granulocytecolony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), growth differentiation factor-9(GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor(HDGF), insulin, insulin-like growth factor (IGF), migration-stimulatingfactor, myostatin (GDF-8), nerve growth factor (NGF) and otherneutrophins, platelet-derived growth factor (PDGF), thrombopoietin(TPO), transforming growth factor alpha (TGF-α), transforming growthfactor beta (TGF-β), tumor necrosis factor-alpha (TNF-α), vascularendothelial growth factor (VEGF), writ signaling pathway agonists,placental growth factor (PlGF), fetal Bovine somatotrophin (FBS),interleukin-1 (IL-1), I.-2, IL-3, IL-4, IL-5, IL-6, IL-7, and the like.In one embodiment, the cell culture media is supplemented with thepoint-of-use addition growth factor insulin. In one embodiment, theconcentration of insulin in the media, i.e., the amount of insulin inthe cell culture media after addition, is from about 0.1 μM to 10 μM.

Buffers are generally known in the art. The invention is not restrictedto any particular buffer or buffers, and any one of ordinary skill inthe art can select an appropriate buffer or buffer system for use with aparticular cell line producing a particular protein. In one embodiment,a point of-use addition buffer is NaHCO3. In another embodiment, thebuffer is HEPES. in other embodiments the point-of-use addition buffercomprises both NaHCO3 and HEPES.

Energy sources for use as a point-of-use addition in cell culture arealso well known in the art. Without limitation, in one embodiment, thepoint-of-use addition energy source is glucose. Given the particular andspecific requirements of a particular cell line and the protein to beproduced, in one embodiment the glucose can be added to a concentrationof about 1 to 20 mM in the media. In some cases, glucose can be added athigh levels of 20 g/L or higher.

Chelators are likewise well known in the art of cell culture and proteinproduction. Tetrasodium EDTA dehydrate and citrate are two commonchelators used in the art, although other chelators may be employed inthe practice of this invention. In one embodiment, a point-of-useaddition chelator is tefrasodium EDTA dihydrate. In one embodiment, apoint-of-use addition chelator is citrate, such as Na₃C₈H₅O₇.

In one embodiment, the cell culture medium may additionally besupplemented with one or more point-of-use addition amino acids as anenergy source, such as e.g., glutamine. In one embodiment, the cellculture media is supplemented with the point-of-use addition glutamineat a final concentration of about 1 mM to 13 mM.

Other point-of-use additions include one or more of various metal salts,such as salts of iron, nickel, zinc and copper. In one embodiment, thecell culture media is supplemented with any one or more of coppersulfate, zinc sulfate, ferric chloride, and nickel sulfate.

In some embodiments, the protein titer yielded from cell culture intaurine supplemented media is at least 4%, at least 5%, at least 6%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least12%, at least 13%, at least 14%, at least 15%, at least 16%, at least17%, at least 18%, at least 19%, at least 20%, at least 21%, at least22% greater, at least 23% greater, at least 24% greater, at least 25%greater, at least 26% greater, at least 27% greater, at least 28%greater or at least 29% greater than the protein titer (yield) fromcells cultured in non-taurine supplemented. In some embodiments, theprotein titer yielded from cells in taurine supplemented media is atleast 2%, at least 3%, at least 4%, or at least 5% greater than theprotein titer (yield) from similar or identical cells cultured innon-taurine supplemented media

In some embodiments, the ammonia accumulation in cell culture isdecreased greater than 4%, greater than 5%, greater than 6%, greaterthan 7%, greater than 8%, greater than 9%, greater than 10%, greaterthan 15%, or greater than 20% in taurine supplemented media compared toceil culture in non-taurine supplemented media.

Protein Production

In addition to taurine supplemented media and methods of culturing cellsin taurine supplemented media, the present invention provides improvedmethods of producing a protein, such as a therapeutically effectiveantibody or other biopharmaceutical drug substance in a cell cultured intaurine supplemented media. The present invention provides a method forproducing therapeutic protein in high yield comprising culturing arecombinant cell line in medium containing taurine, wherein the cellline comprises a stably integrated nucleic acid encoding the therapeuticprotein.

In some embodiments, the titer (yield) of protein by mammalian cellscultured in medium containing taurine (taurine supplemented medium) isat least 100 mg/L, at least 0.5 g/L, at least 1 g/L, at least 1.2 g/L,at least 1.4 g/L, at least 1.6 g/L. at least 1.8 g/L, at least 2 g/L, atleast 2.5 g/L greater than the titer of protein by an identicalmammalian cell cultured in non-taurine supplemented medium.

In some embodiments, the protein production yield or titer, which can beexpressed in grams of protein product per liter of culture medium, fromcells cultured in taurine supplemented medium is at least 100 mg/L, atleast 1 g/L, at least 1.2 g/L, at least 1.4 g/L, at least 1.6 g/L, atleast 1.8 g/L, at least 2 g/L, at least 2.5 g/L, at least 3 g/L, atleast, 3 5 g.L, at least 4 g/L, at least 4 5 g/L, at least 5 g/L, atleast 5.5 g/L, at least 6g/l, at least 6.5 g/L, at least 7 g/L, at least7.5 g/L, at least 8 g/L, at least 8.5 g/L, at least 9g/L, at least 9.5g/L, at least 10 g/L, at least 15 g/L, or at least 20 g/L.

In some embodiments, the protein titer yielded from cells in taurinesupplemented media is at least 2%, at least 3%, at least 4%, at least5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, atleast 21%, at least 22%, at least 23% greater, at least 24% greater, atleast 25% greater, at least 26% greater, at least 27% greater, at least28% greater or at least 29% greater than the protein titer (yield) fromsimilar or identical cells cultured in non-taurine supplemented media.

In some embodiments, the protein product (protein of interest) is anantibody, a human antibody, a humanized antibody, a chimeric antibody, amonoclonal antibody, a multispecific antibody, a bispecific antibody, anantigen binding antibody fragment, a single chain antibody, a diabody,triabody or tetrabody, a Fab fragment or a F(ab′)2 fragment, an IgDantibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgGlantibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, inone embodiment, the antibody is an IgG1 antibody. In one embodiment, theantibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4antibody, in one embodiment, the antibody is a chimeric IgG2/IgG4antibody. In one embodiment, the antibody is a chimeric IgG2/IgG1antibody, in one embodiment, the antibody is a chimeric IgG2/IgG1/IgG4antibody.

In some embodiments, the antibody is selected from the group consistingof an anti-Programmed Cell Death 1 antibody (e.g. an anti-PD1 antibodyas described in U.S. Pat. Appln. Pub. No. US2015/0203579A1), ananti-Programmed Cell Death Ligand-1 (e.g. an anti-PD-L1 antibody asdescribed in in U.S. Pat. Appln Pub No US2015/0203680A1). an anti-Dll4antibody, an anti-Angiopoetin-2 antibody (e.g. an anti-ANG2 antibody asdescribed in U.S. Pat. No 9.402,898), an anti-Angiopoetin-Like 3antibody (e.g. an anti-AngPtl3 antibody as described in U.S. Pat. No.9,018,356), an anti-platelet derived growth factor receptor antibody(e.g. an anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827),an anti-Erb3 antibody, an anti-Prolactin Receptor antibody (e.g.anti-PRLR antibody as described in U.S. Pat. No. 9.302,015), ananti-Complement 5 antibody (e.g. an anti-C5 antibody as described inU.S. Pat Appln. Pub. No US2015/0313194A1), an anti-TNF antibody, ananti-epidermal growth factor receptor antibody (e.g. an anti-EGFRantibody as described in U.S. Pat. No. 9,132,192 or an anti-EGFRvIIIantibody as described in U.S. Pat Appln. Pub. No. US2015/0259423A1). ananti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g. ananti-PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S Pat.Appln. Pub. No. US2014/0044730A1), an anti-Growth And DifferentiationFactor-8 antibody (e.g. an anti-GDF8 antibody, also known asanti-myostatin antibody, as described in U.S. Pat. Nos. 8,871.209 or9,260,515), an anti-Glucagon Receptor (e.g. anti-GCGR antibody asdescribed in U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 orUS2016/0075778A1), an anti-VEGF antibody, an anti-IL1R antibody, aninterleukin 4 receptor antibody (e.g. an anti-IL4R antibody as describedin U.S Pat. Appln. Pub. No US2014/0271681A1 or U.S. Pat. Nos. 8,735,095or 8,945,559), an anti-interleukin 6 receptor antibody (e.g. ananti-IL6R antibody as described in U.S. Pat Nos. 7,582,298, 8,043,617 or9,173,880), an anti-IL1 antibody, an anti-IL2 antibody, an anti-IL3antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g. anti-IL33antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0271658A1 orUS2014/0271642A1), an anti-Respiratory syncytial virus antibody (e.g.anti-RSV antibody as described in U.S. Pat. Appln. Pub No.US2014/0271653A1), an anti-Cluster of differentiation 3 (e.g. ananti-CD3 antibody, as described in U.S. Pat. Appln. Pub. Nos.US2014/0088295A1 and US20150266S66A1, and in U.S. Application No62/222,605), an anti-Cluster of differentiation 20 (e.g. an anti-CD20antibody as described in U.S Pat. Appln. Pub Nos. US2014/0088295A1 andUS20150266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19 antibody,an anti-CD28 antibody, an anti-Cluster of Differentiation-48 (e.g.anti-CD48 antibody as described in U.S. Pat. No. 9,228,014), an anti-Feld1 antibody (e.g. as described in U.S. Pat. No. 9,079,948), ananti-Middle East Respiratory Syndrome virus (e.g. an anti-MERS antibodyas described in U.S Pat. Appln. Pub. No. US2015/0337029A1), ananti-Ebola virus antibody (e.g. as described in U.S. Pat Appln. Pub. No.US2016/0215040), an anti-Zika virus antibody, an anti-LymphocyteActivation Gene 3 antibody (e.g. an anti-FLAG3 antibody, or ananti-CD223 antibody), an anti-Nerve Growth Factor antibody (e.g. ananti-NGF antibody as described in U.S. Pat. Appln. Pub. No.US2016/0017029 and U.S. Pat. Nos. 8,309,088 and 9,353,176) and ananti-Activin A antibody. In some embodiments, the bispecific antibody isselected from the group consisting of an anti-CD3 x anti-CD20 bispecificantibody (as described in U.S. Pat. Appln. Pub. Nos US2014/0088295A1 andUS20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (e.g.,an anti-CD3 x anti-Muc16 bispecific antibody), and an anti -CD3 x anti-Prostate-specific membrane antigen bispecific antibody (e.g., ananti-CD3 x anti-PSMA bispecific antibody). In some embodiments, theprotein of interest is selected from the group consisting of alirocumab,sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab,and rinucumab. All publications mentioned throughout this disclosure areincorporated herein by reference in their entirety.

In some embodiments, the protein of interest is a recombinant proteinthat contains an Fc moiety and another domain, (e.g., an Fc-fusionprotein). In some embodiments, an Fc-fusion protein is a receptorFc-fusion protein, which contains one or more extracellular domain(s) ofa receptor coupled to an Fc moiety. In some embodiments, the Fc moietycomprises a hinge region followed by a CH2 and CH3 domain of an IgG. Insome embodiments, the receptor Fc-fusion protein contains two or moredistinct receptor chains that bind to either a single ligand or multipleligands. For example, an Fc-fusion protein is a TRAP protein, such asfor example an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcPligand binding region fused to the II-1R1 extracellular region fused toFc of hlgG1; see U.S. Pat. No. 6,927,004, which is herein incorporatedby reference in its entirety), or a VEGF trap (e.g., afibercept orziv-aflibercept, which contains the Ig domain 2 of the VEGF receptorFlt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc ofhlgG1; see U.S. Pat. Nos 7,087,411 and 7.279,159). In other embodiments,an Fc-fusion protein is a ScFv-Fc-fusion protein, which contains one ormore of one or more antigen-binding domain(s). such as a variable heavychain fragment and a variable light chain fragment, of an antibodycoupled to an Fc moiety.

The present invention is not limited to any particular type of cell forprotein production. Examples of cell types suitable for proteinproduction include mammalian cells, insect cells, avian cells, bacterialcells, and yeast cells. The cells may be stem cells or recombinant cellstransformed with a vector for recombinant gene expression, or cellstransfected with a virus for producing viral products. The cells maycontain a recombinant heterologous polynucleotide construct that encodesa protein of interest. That construct can be an episome or it can be anelement that is physically integrated into the genome of the cell. Thecells may also produce a protein of interest without having that proteinencoded on a heterologous polypeptide construct. In other words, thecell may naturally encode the protein of interest such as a B-cellproducing an antibody. The cells may also be primary cells, such aschicken embryo cells, or primary cell lines. Examples of useful cellsinclude BSC ceils, LLC-MK cells. CV-1 cells, COS cells, VERO cells, MDBKcells. MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPKcells. PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, chickenembryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 293cells, RK cells, Per C6 cells and CHO cells. In various embodiments, thecell line is a CHO cell derviative, such as CHO-K1, CHO DUX B-11, CHODG-44, Veggie-CHO. GS-CHO, S-CHO, or CHO lec mutant lines.

In one embodiment, the cell, which is a CHO cell, ectopically expressesa protein. In one embodiment, the protein comprises an immunoglobulinheavy chain region, such as a CH1, CH2, or CH3 region. In oneembodiment, the protein comprises a human or rodent immunoglobulin CH2and CH3 region. In one embodiment, the protein comprises a human orrodent immunoglobulin CH1, CH2, and CH3 region. In one embodiment, theprotein composes a hinge region and a CH1, CH2, and CH3 region. In oneembodiment, the protein composes an immunoglobulin heavy chain variabledomain. In one embodiment, the protein comprises an immunoglobulin lightchain variable domain. In one embodiment, the protein comprises animmunoglobulin heavy chain variable domain and an immunoglobulin lightchain variable domain. In one embodiment, the protein is an antibody,such as a human antibody, a rodent antibody, or a chimeric human-rodentantibody (e.g., human/mouse, human/rat, or human hamster).

A production phase can be conducted at any scale of culture, from shakerflasks or wave bags, to one-liter bioreactors, and to large scaleindustrial bioreactors. Likewise a seed train expansion phase can beconducted at any scale of culture, from and shaker flasks or wave bags,to one-liter or larger bioreactors. A large scale process can beconducted in a volume of about 100 liters to 20,000 liters or more. Oneor more of several means may be used to control protein production, suchas temperature shift or chemical induction. A growth phase may occur ata higher temperature than a production phase. For example, a growthphase may occur at a first temperature of about 35° C. to 38° C., and aproduction phase may occur at a second temperature of about 29° C. to37° C, optionally from about 30° C. to 36° C. or from about 30° C. to34° C. In addition, chemical inducers of protein production, such ascaffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, andhexamethylene bisacetamide (HMBA), may be added concurrent with, before,or after a temperature shift. If inducers are added after a temperatureshift, they can be added from one hour to five days after thetemperature shift, such as from one to two days after the temperatureshift. Production cell cultures may be run as continuous feed culturesystem, as in a chemostat (see C Altamirano et al., 2001 supra), oraccording to a fed-batch process (Huang, 2010 supra).

The invention is useful for improving protein production via cellculture processes. The cell lines used in the invention can begenetically engineered to express a polypeptide of commercial orscientific interest. Genetically engineering the cell line involvestransfecting, transforming or transducing the cells with a recombinantpolynucleotide molecule, or otherwise altering (e.g., by homologousrecombination and gene activation or fusion of a recombinant cell with anon-recombinant cell) so as to cause the host cell to express a desiredrecombinant polypeptide. Methods and vectors for genetically engineeringcells or cell lines to express a polypeptide of interest are well knownto those of skill in the art; for example, various techniques areillustrated in Current Protocols in Molecular Biology. Ausubel et al.,eds. (Wiley & Sons. New York, 1988, and quarterly updates): Sambrook etal., Molecular Cloning. A Laboratory Manual (Cold Spring LaboratoryPress, 1989): Kaufman. R. J. Large Scale Mammalian Cell Culture, 1990,pp 15-69. A wide variety of cell lines suitable for growth in cultureare available from the American Type Culture Collection (Manassas, Va.)and commercial vendors. Examples of cell lines commonly used in theindustry include VERO, BHK, HeLa, CVI (including Cos). MDCK, 293, 3T3,myeloma cell lines (e.g., NSO, NSI), PC12, WI38 cells, and Chinesehamster ovary (CHO) cells. CHO cells are widely used for the productionof complex recombinant proteins, such as cytokines, clotting factors,and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al.(1988), J. Biol Chem 263:6352-6362; McKinnon et al (1991), J MolEndocrinol 6:231-239, Wood et al (1990). J Immunol 145:3011-3016). Thedihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub etal. (1980) Proc Natl Acad Sci USA 77: 4216-4220), DXBI 1 and DG-44, aredesirable CHO host cell lines because the efficient DHFR selectable andamplifiable gene expression system allows high level recombinant proteinexpression in these cells (Kaufman RJ. (1990) Meth Enzymol 185:537-566).In addition, these cells are easy to manipulate as adherent orsuspension cultures and exhibit relatively good genetic stability. CHOceils and the proteins recombinantly expressed by them have beenextensively characterized and have been approved for use in clinical andcommercial manufacturing by regulatory agencies .In some embodiments,the CHO cell lines are cell lines as described in U.S. PatentApplication Publication Nos. 2010/0304436 A1, 2009/0162901 A1 and2009/0137416 A1, and U.S. Pat. Nos. 7,455,988 B2. 7,435,553 B2. and7,105,348 B2.

The present invention is not limited in scope by the specificembodiments described herein, which are intended as illustrations ofindividual aspects or embodiments of the invention. Functionallyequivalent methods and components are within the scope of the invention.Various modifications of the invention, in addition to those describedhere, are apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications fall withinthe scope of the invention.

EXAMPLES Example 1: Improved Antibody Titers Due to TauringSupplementation

Example 1A—High-Throughput Shake Flask Culture: 250 mL shake flasks wereinoculated from a seed culture of a monoclonal antibody (Ab1) producingcell line derived from CHO-K1. The inoculated cells were grown at 35.5°C. for seventeen days and fed glucose and other supplemental nutrientsas needed. Cells were grown in chemically defined (hydrolysate-free andserum-free) base media.

Each culture flask was either unsupplemented (Flask 1a), or supplementedwith 1 mM taurine at day 0 (Flask 1b).

TABLE 2 Average 17-Day Antibody Titers (g/L) and Approximate TiterIncrease (%) Relative to Baseline Flask Medium Supplement Ab1 Titer 1aUnsupplemented* 7.3 g/L 1b Taurine 7.9 g/L{circumflex over ( )} 8%*Baseline control for % titer increase; Flask 1b compared to titer inunsupplemented media (Flask 1a). {circumflex over ( )}Difference infinal titer between unsupplemented and supplemented culture isstatistically significant (p < 0.05).

Titer values were calculated from protein harvested on day 17, and arestatistically significant (p<0.05) compared to baselineTaurine-supplemented cultures exhibit an overall 0% increase in finalprotein titer over unsupplemented cultures.

Example 1B—Benchtop-Scale Bioreactors in a similar example, yet on alarger scale of production, 2L bioreactors were inoculated from a seedculture of a monoclonal antibody (Ab2, Ab3 or Ab4) producing cell linederived from CHO-K1. The inoculated cultures were grown at a temperatureof 35.5° C, DO set point of 40.4% and air sparge of 22 ccm for 14 days.Ab2 and Ab3 processes had pH setpoints of 7.0±0.15, while the Ab4process had a pH setpoint of 7.13±0 27. Glucose, antifoam and basal feedwere supplied to the bioreactors as needed. Cultures were grown inunsupplemented medium (Bioreactor 2 a, 3 a, 4 a) or grown in about 1 mMtaurine-supplemented medium (Ab2 and Ab3) or about 3 mMtaurine-supplemented medium (Ab4). added on day 0 of production(Bioreactors 2 b, 3 b and 4 b, respectively).

Antibody yield (titer) was 6.4 g/L for Ab2-producing cells, yet thecells grown with taurine yielded 8 g/L protein The 24% increase in titercompared to cells grown without taurine supplementation is statisticallysignificant (p<0.05). The resulting final titers for Ab3-producingcultures and Ab4-producing cultures are also significantly higher(p<0.05) after 14 days (11% and 20% respectively), compared tonon-taurine supplemented cultures. See Table 3.

TABLE 3 Average 14-Day Antibody Titers (g/L) and Approximate TiterIncrease (%) Relative to Baseline Medium Bio- Ab2 Bio- Ab3 Bid- Ab4Supplement reactor# Titer reactor# Titer reactor# Titer Unsupplemented*2a 6.4 g/L 3a 6.6 g/L 4a 4.4 g/L Taurine 2b   8 g/L{circumflex over ( )}24% 3b 7.3 g/L{circumflex over ( )} 11% 4b 5.3 g/L{circumflex over ( )}20% *Unsupplemented medium is the baseline control for % titer increase,where Bioreactors 2b, 3b, or 4b % titer increase is compared to titer inunsupplemented media (Bioreactors 2a, 3a, or 4a, respectively).{circumflex over ( )}Differences in final titer between supplemented andunsupplemented cultures are statistically significant (p < 0.05).

A timecourse with regard to protein titer was plotted for theAb3-producing cell culture and significant improvement in protein titerdue to taurine supplementation was observed at each day of culture,starting at day 6.

TABLE 4 Antibody Titer (g/L) Improvement Due to Taurine Supplementationat Representative Timepoints Media Ab3 Titer Ab3 Titer Ab3 TiterBioreactor Supplement at day 6 at day 9 at day 14 3a Unsupplemented* 1.7g/L 4.1 g/L 6.6 g/L 3b Taurine 1.9 g/L 12% 5.3 g/L{circumflex over ( )}29% 7.3 g/L{circumflex over ( )} 11% *Approximate increase (%) comparedto unsupplemented medium collected on same day {circumflex over( )}Increase in titer with taurine supplementation is statisticallysignificant (p < 0.05) compared to unsupplemented culture.

A significant improvement in titer could be seen in production cultureas early as day 6 (12% increase compared to the same culture withouttaurine supplementation). See also the FIGURE. The maximum difference inthis timecourse was seen at day 9 (significant (p<0.05) increase of 29%in Bioreactor 3b compared to 3a), and significant (p<0.05) titerincrease of 11% was observed on the final day (14) of culture.

The protein production benefits of taurine-supplementation are observedacross different scales (Example 1A and 1B) and different cell lines(Example 1B).

Example 2: Consistent Productivity with Varying Taurine Concentrationsin a High-Throughput Shake Flask Culture

Consistency of the protein titer was tested by varying the amount oftaurine added to the culture at production day 0.250 mL shake flaskswere inoculated from a seed culture of a monoclonal antibody (Ab1)producing cell line derived from CHO-K1. The inoculated cells were grownat 35.5° C. for fourteen days and fed glucose and other supplementalnutrients as needed. Cells were grown in chemically defined(hydrolysate-free and serum-free) base media.

Each culture contained either no taurine (unsupplemented) or taurine at0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM, 3 mM, 5 mM, 7.5 mM or 10 mMconcentrations.

TABLE 5 Average 14-Day Antibody Titers (g/L) for Cultures Supplementedwith 0.1 to 10 mM Taurine Shake Media Ab1 Flask Supplement Titer 5aUnsupplemented* 6.5 g/L 5b 0.1 mM Taurine^(#) 6.7 g/L 3% 5c 0.3 mMTaurine{circumflex over ( )} 6.8 g/L 5% 5d 0.5 mM Taurine{circumflexover ( )} 6.9 g/L 6% 5e 0.7 mM Taurine{circumflex over ( )} 7.0 g/L 8%5f   1 mM Taurine{circumflex over ( )} 7.0 g/L 8% 5g   5 mMTaurine{circumflex over ( )} 7.1 g/L 9% 5h 7.5 mM Taurine{circumflexover ( )} 7.1 g/L 9% 5i  10 mM Taurine{circumflex over ( )} 7.1 g/L 9%*Unsupplemented medium is the baseline control for % titer increase.^(#)Difference in final titer is statisically significance compared tounsupplemented control (p < 0.1). {circumflex over ( )}Difference infinal titer is statistically significance compared to unsupplementedcontrol (p < 0.05).

It is shown that varying the amount of taurine-supplementationconsistently produces high liters, when taurine is supplemented in arange of at least 0.1 mM to 10 mM. Final titers for the taurinesupplemented conditions were statistically different from theunsupplemented condition. For 0.1 mM taurine, p<0.1 while p<0.05 for 0.3mM to 10 mM taurine.

Example 3: Testing Varying Taurine Feeding Schedules in aHigh-Throughput Shake Flask Culture Example 3A: Addition of TaurineDuring Seed Train Expansion Phase

The benefits of adding taurine to the culture during the expansion seedtrain phase were assessed in the high-throughput shake flask model. Inflask 6a (Table 6), the Ab1-producing CHO cells were thawed inchemically defined (hydrolysate-free and serum-free) base mediasupplemented with 1 mM taurine Taurine concentration of basal medium wasmaintained at 1 mM throughout the expansion phase. During production,the culture basal medium was supplemented with 1 mM taurine on day 0.Glucose and nutrient basal feed were supplied as needed during the 17day production.

Flask 6 b cells grew in taurine-free (unsupplemented) chemically defined(hydrolysate-free and serum-free) basal medium throughout the seed trainexpansion phase. At day 0 of production, the culture basal medium wassupplemented with 1 mM taurine. For the 17 day production, glucose andnutrient basal feeds were supplied as needed.

TABLE 6 Average 17-Day antibody Titers for Cultures Supplemented with 1mM Taurine during Different Phases of the Process Shake Addition of 1 mMFlask Taurine Ab1 Titer 6a Seed Train and 7.7 g/L Production Phases 6bOnly Production Phase 7.8 g/L Differences in final titer (day 17) arenot statisically significant (p > 0.1).

Final (day 17) titer values for both conditions (taurine supplementationin production only or seed train and production combined) are similar.Resulting titers are not statistically significant (p>0.1). The benefitof adding taurine in the seed train expansion phase is analogous tosupplementing taurine h the production phase.

EXAMPLE 3B: Varying Taurine Feeding Schedules During Production Phase:To determine whether varying standard feeding schedules of taurine hadany effect on the protein titer for taurine-supplemented cultures,additional experiments were conducted in analogous shake flask culturesgrowing Ab1-producing CHO cells. The cells were subjected to varyingculture conditions similar to Example 2, where the feeding schedule wasthe same as before, glucose/nutrient basal feed was added as needed.

Ab1-producing cultures were supplemented with a total of 5 mM taurine.Similar productivity (7.1 g/L, 6.8 g/L and 7.0 g/L) is observed invarying taurine feeding schedules. Titer values as compared in thisexperiment are not statistically different (p>0.1) (see Table 7).

TABLE 7 Average 14-Day Antibody Titers (g/L) for Cultures Supplementedwith 5 mM Taurine with Varying Schedules Ab1 Taurine Schedule Day 14Titer 5 mM day 0 7.1 g/L 1 mM days 0, 3, 5, 7, 10 6.8 g/L (5 mM total) 1mM day 0: 7.0 g/L 2 mM days 7, 10 (5 mM total) Differences between day14 titer values are not staistically significant (p > 0.1).

The feeding schedules of taurine do not have any negative effect, noralter the outcome where taurine-supplementation is beneficial to productyield. Thus, taurine supplementation may be added once at day 0, oradded on subsequent days of the production phase, or may be added atmultiple intervals during the production phase.

Example 4: Measuring Ammonia Byproduct in a High-Throughput Shake FlaskCulture

Ammonia by-product was measured following 14-day cultures conducted inan analogous manner as Example 2 for taurine-supplemented cultures ofAb1-producing CHO cells. The cells were subjected to varying cultureconditions similar to above where glucose/nutrient base feed was addedas needed.

TABLE 8 Average 17-Day Ammonia (mM) and Decrease (%) Ab1 MediaSupplement Ammonia Unsupplemented* 2.56 mM Taurine 1.73 mM{circumflexover ( )} −32% *Baseline control for % ammonia decrease; taurinesupplemented condition compared to ammonia in unsupplemented medium.{circumflex over ( )}Decrease in ammonia concentration is statisticallysignificant (p < 0.1).

In the Ab1-producing cells, taurine supplementation in the mediumsupports a heathy. sustainable culture where the ammonia by-product hasbeen reduced by 32% The decrease in ammonia concentration from taurinesupplementation is statistically significant (p<0.1).

The present invention may be embodied in other specific embodiments.

What is claimed is
 1. A method for cultivating a eukaryotic cellexpressing aflibercept, comprising: (a) providing a base cell culturemedium supplemented about 0.1 mM to about 10 mM L-taurine; and (b)propagating or maintaining the eukaryotic cell in said base cell culturemedium to form a cell culture.
 2. The method of claim 1, wherein thebase cell medium is chemically defined.
 3. The method of claim 2,wherein the base cell medium is serum and hydrolysate free.
 4. Themethod of claim 1, wherein the base cell medium is supplemented withornithine, putrescine or a combination thereof at (b).
 5. The method ofclaim 1, further comprising a growth phase prior to step (b), whereinthe growth phase comprises propagating or maintaining the eukaryoticcells in a base cell culture medium that is not supplemented withL-taurine.
 6. The method of claim 1, further comprising a growth phaseprior to step (b), wherein the growth phase comprises propagating ormaintaining the eukaryotic cells in a base cell culture medium that issupplemented with about 0.1 mM to about 10 mM L-taurine.
 7. The methodof claim 1, wherein the taurine supplement of is provided at least once,at least twice, at least 3 times, at least 4 times, or at least 5 timesduring step (b).
 8. The method of claim 1, wherein the taurinesupplement of (b) is provided on each day.
 9. The method of claim 1,wherein the base cell culture medium comprises a mixture of amino acidsselected from the group consisting of arginine, histidine, lysine,aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine,cysteine, glycine, proline, alanine, valine, isoleucine, leucine,methionine, phenylalanine, tyrosine, and tryptophan.
 10. The method ofclaim 1, wherein the base cell culture medium comprises one or morefatty acids.
 11. The method of claim 10, wherein the one or more fattyacids are selected from the group consisting of linoleic acid, linolenicacid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidicacid, arachidonic acid, lauric acid, hehenic acid, decanoic acid,dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, andoctanoic acid.
 12. The method of claim 1, wherein the base cell culturemedium comprises vitamins and cofactors selected from the groupconsisting of biotin, D-calcium pantothenate, choline chloride, folicacid, 1-inositol, nicotinamide, pyridoxine HCI, riboflavin, thiamine.HCI, and vitamin B12.
 13. The method of claim 1, wherein the base cellculture medium comprises a mixture of nucleosides.
 14. The method ofclaim 13, wherein the mixture of nucleosides comprises one or more ofadenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.15. The method of claim 1, wherein the base cell culture mediumcomprises one or more divalent cations.
 16. The method of claim 15,wherein the one or more divalent cations comprise Ca2+, Mg2+, or both.17. The method of claim 1, wherein the eukaryotic cell is selected fromthe group consisting of a mammalian cell, an avian cell, an insect cell,and a yeast cell.
 18. The method of claim 17, wherein the mammalian cellis selected from the group consisting of a CHO cell, a COS cell, aretinal cell, a Vero cell, a CV-1 cell, a kidney cell, a HeLa cell, aHepG2 cell, a W138 cell, a MRC 5 cell, a Colo25 cell, a HB 8065 cell, aHL-60 cell, a lymphocyte cell, a A431 cell, a U937 cell, a 3T3 cell, anL cell, a C127 cell, an. SP2/0 cell, an NS-0 cell, an MMT cell, a PER.C6cell, a stem cell, a tumor cell, and a derivative thereof.
 19. Themethod of claim 17, wherein the mammalian cell is a CHO cell, HEK293cell, or BHK cell.
 20. A method for producing aflibercept comprising thesteps of: (a) introducing a nucleic acid comprising a nucleotidesequence encoding aflibercept into a cell; (b) selecting the cellexpressing the aflibercept; (c) culturing the selected cell in a cellculture medium comprising about 0.1 mM to about 10 mM L-taurine, therebyproducing a population of cells expressing aflibercept, wherein theaflibercept is secreted into the medium; and (d) harvesting theaflibercept.
 21. The method of claim 20, wherein the titer of theaflibercept produced by supplementing the cell culture medium withL-taurine is increased when compared to the titer of a comparatorcontrol culture whose cell culture medium has not been supplemented withL-taurine.
 22. The method of claim 21, wherein the titer of theaflibercept is at least 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 20%higher than the titer of the comparator control culture.
 23. The methodof claim 20, wherein the population of cells is capable of at least 3%higher protein yield of aflibercept compared to a population of cellsexpressing aflibercept in a cell culture medium containing less than 0.1mM L-taurine.
 24. The method of claim 20, wherein the population ofcells is capable of at least 8% higher protein yield of afliberceptcompared to cells expressing aflibercept in a cell culture mediumcontaining less than 0.1 mM L-taurine.
 25. The method of claim 20,wherein producing aflibercept by culturing the population of cells in acell culture medium with L-taurine is capable of increasing proteinyield by at least 0.1 g/L, at least 0.5 g/L, at least 1 g/L, at least 1.2 g/L, at least 1 .4 g/L, at least 1 .6 g/L, at least 1 .8 g/L, atleast 2 g/L, at least 2.2 g/L, at least 2.4 g/L, or at least 2.5 g/Lwhen compared to culturing the population of cells in a similar cellculture medium that contains less than 0.1 mM taurine.
 26. The method ofclaim 20, wherein the cell is a CHO cell, HEK293 cell or BHK cell. 27.The method of claim 20, comprising culturing the population of cells inthe cell culture medium supplemented with L-taurine at (c) for at least6 days.
 28. The method of claim 20, wherein the nucleic acid encodingaflibercept is stably integrated in the cell.
 29. The method of claim20, wherein supplementing the cell culture medium with L-taurine iscapable of decreasing ammonia accumulation by at least 3%, or at least8%, when compared to a similar cell culture medium that contains lessthan 0.1 mM taurine.
 30. The method of claim 20, comprising the step ofadding one or snore point-of-use additions to the cell culture medium.